Vitamin D is a group of prohormones. The two major forms of vitamin D are vitamin D2 and vitamin D3. Vitamin D is metabolized to 25-hydroxy vitamin D (calcidiol) in the kidney and 1,25-dihydroxy vitamin D (calcitriol) in the liver.
Vitamin D facilitates the flow of calcium into the bloodstream. The flow of calcium into the bloodstream is important for the mineralization of bone and the prevention of hypocalcemic tetany.
Vitamin D assays have become increasingly popular in North America. The development of highly specific anti-vitamin D monoclonal antibodies for vitamin D assays, however, has proven challenging. Various animal immunization protocols have been carried out in efforts to produce monoclonal antibodies specific for 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3. The combined measurement of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 (collectively, 25-hydroxy vitamin D) is considered to be the best indicator of the total amount of vitamin D in the body.
Vitamin D hormones have essential roles in human health mediated by intracellular vitamin D receptors (VDR). For instance, the vitamin D hormones regulate blood calcium levels by controlling the absorption of dietary calcium by the small intestine and the reabsorption of calcium by the kidneys. Excessive hormone levels can lead to abnormally elevated urine calcium (hypercalciuria), blood calcium (hypercalcemia) and blood phosphorus (hyperphosphatemia). Vitamin D hormones also participate in the regulation of cellular differentiation and growth, parathyroid hormone (PTH) secretion by the parathyroid glands, and normal bone formation and metabolism. Furthermore, vitamin D hormones are required for the normal functioning of the musculoskeletal, immune and rennin/angiotensin systems. Numerous other roles for the vitamin D hormones have been identified and proposed based on the documented presence of intracellular VDR in nearly every human tissue.
Vitamin D binding protein (DBP) is a triple-domain, monomeric glycoprotein that occurs naturally in mammals. It is a member of the albumin, α-fetoprotein, and α-albumin/afamin gene family. Initially, DBP was named group-specific component of serum (Gc-globulin). The majority of serum DBP is expressed and secreted from the liver. It binds, solubilizes, and transports vitamin D in blood with binding affinities ranging from about 2 nM to about 20 nM (White et al., Trends Endocrinol. Metab. 11: 320-327 (2000); and Cooke et al., “Vitamin D binding protein,” In: Vitamin D, Feldman et al., eds., Academic Press (1997), pages 87-101). DBP also binds to G-actin (Speeckaert et al., Clinica Chimica Acta 372: 33-42 (2006)), which is released from cells to the blood upon injury, with a binding affinity of about 10 nM, and fatty acids (Calvo, Biochem. Biophys. Res. Comm 163: 14-17 (1989)). Human DBP has dominant alleles, namely GC1F, GC1S and GC2, and over 124 other alleles (Cleve et al., Vox Sang. 54: 215-225 (1988)). The normal adult level of DBP is 4-8 μM (200-400 mg/L), which is about 20-100 fold higher than its ligands, namely vitamin D and its metabolites (White et al. (2000), supra). Structural and biochemical characterization has revealed that domain I of DBP (amino acids 1-191) binds vitamin D (specifically amino acids 35-49 of domain I (confirmed by crystal structure)), whereas domain III (amino acids 379-458) binds G-actin (specifically amino acids 373-403 of domain III (crystal structure indicates actin interacts with distinct amino acid sequences in all of the three domains)) (Haddad et al., Biochemistry 31: 7174-7181 (1992); Swamy et al., Biochem. 36: 7432-7436 (1997); Verboven et al., Nature Structural Biology 9: 131-136 (2002); Swamy et al., Arch. Biochem. Biophys. 402: 14-23 (2002); Head et al., Biochem. 41: 9015-9020 (2002); and Otterbein et al., PNAS USA 99: 8003-8008 (2002)). Human DBP has 458 amino acids (after cleavage of a 16-amino acid signal peptide), a high cysteine content (28 cysteines, all in disulfide form), and a molecular weight of 52 kD and migrates at 58 kD on an SDS-PAGE gel. Analysis of truncated proteins and overlapping peptides has indicated that cell binding is mediated by amino acids 150-172 of domain I and amino acids 379-402 of domain III (Zhang et al. Biochim Biophys. Acta 1803: 623-629 (2010)).
Competitive protein-binding assays for 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D were introduced more than three decades ago, and were based on assessing circulating 25-hydroxy vitamin D or 1,25 dihydroxy-vitamin D using the DBP or chick intestinal receptor, respectively, as the primary binding agents (Hollis, Nutrition Reviews 65(8): S87-S90 (August 2007 (II)). Both assays employed 3H-labeled compounds as reporters, and, while valid, they were cumbersome. Since then, assays for both metabolites have advanced and include the use of various methods, such as radioimmunoassay, enzyme-linked immunoassay, high-performance liquid chromatography, liquid chromatography coupled with mass spectrometry, and random access automated assay based on chemiluminescence. U.S. Pat. No. 7,087,395 discloses the use of a releasing composition including cyclodextrin, sodium salicylate, and sodium hydroxide, 25-hydroxy vitamin D coupled to a solid phase, DBP, and labeled anti-DBP antibody, which produces a detectable signal in the presence of 25-hydroxy vitamin D, to determine the amount of 25-hydroxy vitamin D in a sample. U.S. Pat. No. 7,482,162 B discloses adding a non-competitive disaplacement agent comprising 8-anilino-1-napthalene-sulfonic acid ammonium salt, 3-(acetonylbenzyl)-4-hydroxycoumarin and a water-miscible solvent, to a serum or plasma sample to separate any vitamin D metabolite in the sample from protein to which the vitamin D metabolite is bound, such that any separated vitamin D metabolite can be captured, contacting the sample with a support having immobilized thereon a binding factor to capture any vitamin D metabolite separated from protein, and measuring the captured vitamin D metabolite.
The present disclosure seeks to provide a quantitative vitamin D assay using a mutated, truncated DBP fused to human IgG constant fragment (Fc) that binds specifically to 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3. This and other objects and advantages, as well as inventive features, will become apparent from the detailed description provided herein.